ΦC31 integrase mediates efficient cassette exchange in the zebrafish germline

Abstract

Site-specific recombinases (SSRs) are powerful tools for genome manipulation, used in diverse organisms including Drosophila melanogaster, mouse, Arabidopsis, zebrafish, and human cultured cells. The integrase from the bacteriophage ΦC31 belongs to the large serine family of integrases, and in contrast to other widely used SSRs such as Cre and Flp, recombination is directional and therefore irreversible. We have developed a vector system for recombinase-mediated cassette exchange (RMCE) in the zebrafish, allowing swapping of the coding sequence in an integrated transgene. Utilizing codon-optimized ΦC31 integrase RNA bearing the 3'UTR from the nanos1 gene, we replaced the egfp coding sequence of an integrated reporter transgene with mCherry coding sequence. Recombination was achieved at high efficiency in both somatic cells and in the germline. We demonstrate an effective approach to RMCE, increasing the repertoire of tools available to manipulate the zebrafish genome.

Figure 1. Strategy for recombinase–mediated cassette exchange

Enhancer sequences are PCR-amplified and cloned into the attP-cFos-egfp vector via Gateway^™ recombination to generate the target transgene, which is introduced into the germline through Tol2–mediated transgenesis. The donor plasmid, containing the mCherry (mCh) coding sequence flanked by inverted attB sites, is injected into transgenic embryos together with ΦC31o RNA. The expected recombination event will bring mCh expression under control of the specific enhancer sequence.

Figure 2. In vivo activity of ΦC31 integrase in somatic and germ cells

A) Embryos injected with the Sox10:egfp enhancer analysis vector and mCh donor plasmid, together with RNAs encoding Tol2 transposase and ΦC31o integrase, show mCh+ cells in the same tissues as the GFP+ cells seen with injection of the analysis vector alone. Flourescently labeled cells are detected in tissues that are typically positive using the Sox10 enhancer, including the oligodendrocytes of the spinal cord (arrowheads) and glia associated with the lateral line nerve (arrow). B–D) Embryos carrying an integrated egfp transgene with an enhancer from the ACAN gene (A) were injected with the donor plasmid and ΦC31o RNA. Injected embryos display mCh+ cells overlapping the Aggrecan pattern of expression in the notochord at 1 dpf (B), demonstrating appropriate recombination in somatic cells. E–L) ΦC31o mediates recombination in germ cell precursors. Embryos carrying the integrated Sox10:egfp transgene were injected with the mCh targeting plasmid and ΦC31o RNA, and the resulting fish raised to adulthood. In the progeny of some injected fish, mCh expression (G, H) replaced the specific egfp expression pattern (I, J), indicative of RMCE in germ cells of the injected fish. Fish carrying an integrated transgene with an enhancer from TWIST1 were similarly injected with targeting plasmid and ΦC31o RNA; from one founder, embryos with mCh expression in the complete pattern were obtained (I, J), as well as progeny with the original egfp expression (K, L).

Figure 3. Recombination creates hybrid attR site at integrated locus

A) The position of primers used for validation of the recombination event is indicated, on the original egfp transgene and on the locus after successful RCME with the mCh targeting plasmid. B) PCR genotyping: Lane 1, DNA ladder; 2, GFP+ embryo, egfpF and Tol2 primers; 3, mCh+ embryo, egfpF and Tol2 primers; 4, mCh+ embryo, mChF and Tol2 primers; 5, GFP+ embryo, mChF and Tol2 primers. DNA from the mCh+ embryo was also amplified with the mChR and Sox10 primers, and gave the expected size band (not shown). C) Shown are the sequences of the minimal attP site, on the target transgene; the attB site, on the donor plasmid; and the hybrid attR site created at the integrated locus by ΦC31 recombination. Note the underlined triplet in all three sequences, the actual site of recombination. D) Sequence traces from genotyping. From the GFP+ embryo (top) the original attP site was recovered; from the mCh+ embryo, sequence of the hybrid attR site was obtained on both the 3′ (middle) and 5′ (bottom) ends of the mCherry cassette.